Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411

ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 399-411 http://www.ijcmas.com

Original Research Article The Sensitivity of Escherichia coli to Extracts of fragrans, Combretum micranthum and Combretum molle Locally Used in the Treatment of Diarrheal Diseases in the Far-North Region of Cameroon

Toua Vroumsia1*, Pierre Saotoing1, Amadou Dawé2, Moussa Djaouda1, Mbende Ekaney1 and Beltin Abeng Mua1

1Department of Life and Earth Sciences, The University of Maroua Higher Teachers Training College, P. O. Box 55 Maroua, Cameroon 2Department of Chemistry, The University of Maroua Higher Teachers Training College, P. O. Box 55 Maroua, Cameroon *Corresponding author

A B S T R A C T

This study was undertaken to determine the sensitivity of Escherichia coli culture to extracts of three locally used antibacterial (Combretum molle, Combretum fragrans and Combretum micranthum) and to identify the phytochemicals present in these plants. Acetone was used as the solvent for the extraction of the . K e y w o r d s The sensitivity of Escherichia coli culture to the plants extracts was determined on Mueller Hinton Agar, following the agar well diffusion technique. Acetone was Combretum, used as a negative control and Ciprofloxacin as positive control. The Minimum extracts, Inhibitory Concentration of the plant extracts was determined by a modified broth Sensitivity, dilution method. Phytochemical test was done by detecting major colour changes. Escherichia coli, Results indicated that all tested plants extracts, as well as Ciprofloxacin, Inhibition zone, demonstrated antibacterial activity, with diameters of inhibition zones ranging from Minimum 8 ± 0.35 to 36 ± 1.80 mm, while acetone had no activity. Combretum molle and C. Inhibitory fragrans had the greatest inhibitory activity against E. coli. The found MIC varied Concentration from 0.625 to 2.50 mg/ml. Phytochemical tests showed that the plants contained tannins, proteins, flavonoids, phenols, coumarines and glycosides. These results justify the use of the tested plants as antibacterial by the population. Further studies, involving the plants extracts antimicrobial activities against other micro- organisms and their in vivo toxicity and mechanism of action are needed.

Introduction

Diarrhoeal diseases are one of the foremost diarrhoea is prevalent in developing public health problems worldwide. In the countries, particularly in tropical regions 21st century, diarrhoeal diseases continue to (Pickering, 2004). Enteric pathogens are the be a major cause of morbidity and mortality most frequent causes of diarrhoea illness, worldwide (O Ryan et al., 2005). Recurrent which account for an annual mortality rate

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 of 3 million and an estimated 4 billion reason why scientific research in the field of infections worldwide (Talaro, 2005). Almost traditional medicinal plants has to be half of the world s population suffer from intensified in order to find alternative diseases associated with insufficient or antimicrobial drugs. Numerous studies have contaminated water and is at risk from identified active compounds within herbal waterborne and foodborne diseases, of plants that are effective (Basile et which diarrheal diseases are the most deadly al., 2000) and some traditional remedies (Sindiga et al., 1995). have already produced compounds that are effective against -resistant strains The pathogenic bacteria most commonly of bacteria (Koné et al., 2004). associated with endemic forms of diarrhoea are diarrheagenic Escherichia coli, The genus Combretum includes almost 400 Salmonella spp, Shigella, Vibrio cholerae, species found all across Africa, many of Aeromonas and Pleisomonas spp (Mamatha, which are widely used in African traditional 2006). Diarrhoea is also caused by other medicine (Van Wyk and Gericke, 2000). agents like viruses and parasites (Palombo, Several species of the genus have been 2006). reported for their biological activities. Different extracts (ethanol, chloroform, Antibiotic resistance is a major clinical methanol or water) of C. micranthum cause of concern in treating infections antibacterial activity against a number of caused by microorganisms (Acharyya et al., microorganisms such as Pseudomonas 2009). For instance, E. coli has showed aeruginosa, Staphylococcus aureus, resistance to the common cheap antibiotics, Salmonella species, Streptococcus species, notably Trimethoprim-Sulphamethoxazole, Proteus vulgaris, Klebsiella species, Sarcina Kanamycin and Gentamycin (Bebora et al., lutea, Micrococcus luteus and Bacillus 1994). As resistance to old antibiotics subtilis was noted (Neuwinger, 2000). spreads, the development of new Antifungal activity against Candida antimicrobial agents has to be expedited if albicans, antiviral activity against Herpes the problem is to be contained. However, the simplex 1 and 2, antimalarial activity against past record of rapid, wide spread emergence Plasmodium falciparum and antidiabetic of resistance to newly introduced activity was also reported (Masoko et al., antimicrobial agents indicates that even new 2007). Extracts of C. erythrophyllum have families of antimicrobial agents will have a shown antibacterial activity at different short life expectancy (Coates et al., 2002). doses against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus and Medicinal plants are effective in the Enterococcus faecalis (Martini and Eloff, treatment of infectious diseases, while 1998). simultaneously mitigating many of the side effects that are often associated with Phytochemical studies carried out in the synthetic drugs (Koné et al., 2004). Multiple genus Combretum have demonstrated the drug resistance has developed due to the occurrence of many classes of constituents, indiscriminate use of synthetic antimicrobial primarily terpenoids (mainly triterpenes) and drugs (Davis, 1994). In addition, bacteria phenolic compounds (flavonoids, have evolved numerous defences against the stilbenoids, phenanthrenes), among others antimicrobials (Ahmad and Aqil, 2006). A (de Morais Lima et al., 2012). combination of these factors is the more

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As the plants of Combretum genus are they were ground into powder form using a locally and widely used in the treatment of grinder (MK 10-525-B). diarrheal diseases in the Far-North region of Cameroon, it becomes necessary to verify Extraction the scientific basis of this use and to have an idea on the phytochemical composition of All chemicals used in this study were the plants. purchased from Sigma-Aldrich chemicals Co (St. Louis, MO, USA) and were of The general objective of this study is to analytic reagent grade. The powdered plant evaluate the sensitivity of E. coli to extracts materials were soaked in acetone for 48 of the leaves of Combretum molle, hours with intermittent shaking to allow the Combretum fragrans and Combretum active phytochemicals to dislodge in the micranthum. To attempt this, the following solvent. The acetone soaked plant extracts specific objectives are focused: investigation were then filtered by the use of a Whatman of E. coli susceptibility to extracts of the No.1 filter paper and the filtrates evaporated plants leaves, finding of the minimum by using a rotary evaporator (W.2000 inhibitory concentration of the leaves Heidolph, Germany) set at 72oC to remove extracts and screening of the leaves extracts, the excess solvent. The extracts were stored in order to identify the phytochemical in air tight containers in the laboratory until compounds present in these extracts. when they were used.

Materials and methods Antimicrobial assays

Description of the study area Test cultures

The Far-North region is the northern most Bacteria strain of E. coli was isolated from constituent region of the Republic of human faecal matter from patients who Cameroon. It borders the North region to the presented diarrhoea at the Garoua Annex of south, Chad to the east and Nigeria to the the Pasteur Centre of Cameroon. This o west. It is located on latitude 11°00N and bacteria isolate was maintained at 4 C on longitude 14°30E and has a Sudanno- nutrient agar slants until when it was used. sahalian climate, with steppes and savannah vegetation. The annual rainfall is about 400- Preparation of the Mueller Hinton Agar 1500 mm and the annual average (MHA) culture Medium temperature is 28°C. The preparation was done following the Collection of plants materials manufacturer instructions. Generally, 7.6 g of the MHA was added to 200 ml of distilled Fresh plant leaves of Combretum molle R. water and was brought to boil using a Br. ex G. Don, Combretum fragrans F. Bunsen burner. The medium was, later on, Hoffm. and Combretum micranthum G. Don sterilized by autoclaving at 1210C for 15 were harvested in Ngassa, a small village minutes. It was then removed and placed in about a few kilometres from Mindif, in the Petri dishes in a homogenous manner and Far-North Region of Cameroon. The plant allowed to solidify. The Petri dishes leaves were washed and dried at ambient containing agar were then stored in a temperature under shade after collection refrigerator to be used the following day. until they were dry. For laboratory analysis,

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Determination of diameters of growth The medium was then poured into sterile inhibition zones Petri dishes and allowed to set. The surface of the medium was allowed to dry under The Agar Diffusion Method, according to laminar air flow before inoculating with 48 Collins et al. (1995) was used. 0.05 g of h bacterial cultures adjusted to a density of each plant extract was reconstituted in 2.5 0.5 McFarland. Three series of other Petri ml of acetone, leading to a test concentration dishes were used as controls. One for of 20 mg/ml. One colony of an 18 h sterility control containing only 20 ml of conserved bacteria (E. coli) culture was sterilized molten MHA, one containing only dissolved in 2 ml of distilled water in a test 20 ml of contaminated MHA for growth tube and adjusted to a density of 0.5 6 control and one containing only 18 ml of McFarland (approximately 10 CFU/ml) and MHA and 2 ml of acetone, as negative inoculated in prepared MHA plates. control, The Petri dishes were later incubated at 37° C for up to 48 h after which Wells of 6 mm in diameter were made in the they were examined for the presence or agar with a sterile stainless steel cork borer absence of growth (Adesokan et al., 2007). and 0.05 ml of the extracts was added to The MIC was taken as the lowest each well. Controls were comprised of concentration that prevented the growth of extraction solvent acetone (Negative the test microorganism. control) and Ciprofloxacin 5 µg standard disk (Positive control). The agar plates were Qualitative methods of phytochemical incubated at 37° C for 48 h. The different screening zones of inhibition around each plant extract and the controls were recorded. The The extracts of each plant were diameters of these zones of inhibition, analyzed for alkaloids, flavonoids, around the wells containing the extract, were glycosides, phenols, saponins, tannins, taken to indicate the antibacterial activity of quinones, cardiac glycosides, coumarines, the plant extracts against the test organism aromatic amino acids, phytosterols (CASFM, 2014). (triterpenoides), proteins and carbohydrates according to Harborne (1998), with slight Determination of Minimum Inhibitory modifications. Concentration (MIC) Detection of alkaloids The estimation of the MIC of the crude extracts was carried out using a modified About 50 mg of each plant extract were Broth Dilution Technique (Akinpelu and stirred with 3 ml of dilute hydrochloric acid Kolawole, 2004). Two-fold dilutions of each and filtered thoroughly. The Dragendorff of the plants extracts were made, giving test was used. To a 1 ml of filtrate, 2 ml of concentrations of 50, 25, 12.5, 6.25, 3.13, Dragendorff reagent are added. A prominent 1.56 and 0.78 mg/l. Then, two aliquots (2 yellow precipitate confirms the test as ml) of different concentrations of the two- positive. fold dilutions of each crude extract prepared was added to 18 ml of pre-sterilized molten Detection of carbohydrates MHA in test tubes, at temperature of 40°C and mixed to give final concentrations of 10, The Benedict s test was used to point out the 5, 2.50, 1.25, 0.625, 0.313, 0.156, and 0.078 presence of carbohydrates. To 0.5 ml of mg/ml.

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 each plant extract, 0.5 ml of Benedict The formation of blue-green colour reagent was added. The mixture was heated indicated the presence of triterpenoides and on a boiling water bath for 2 min. A red phytosteroids. precipitates indicated presence of sugar.

Detection of glycosides Detection of tannins

To test for the presence of glycosides, The Ferric chloride test was used to test for Legal s test was used. To 2 ml of each plant the presence of tannins. Each extract (5 mg) extract, 3 ml of chloroform and ammonium was dissolved in 5 ml of distilled water and solution (10%) were added. Formation of few drops of 5% ferric chloride solution pink colour indicated the presence of were added. The formation of blue-green glycosides. colour indicated the presence of tannins.

Detection of proteins Detection of phenols

The plant extracts were, each, dissolved in Phenols were tested using the Lead acetate 10 ml of distilled water and filtered through test. Each plant extract (5 mg) was dissolved Whatman No.1 filter paper. Millon test was in distilled water and 3 ml of 5 % lead used to test for proteins. To 2 ml of filtrate, acetate solution was added. A bulky white few drops of Millon reagent were added. A precipitates indicated the presence of white precipitates indicated presence of phenols. proteins. The Biuret test was also used to detect the presence of proteins as follows. Detection of flavonoids An aliquot of 2 ml of filtrate was treated with two drops of 2% copper sulphate An aqueous solution of the extracts was solution. To this, 1 ml of ethanol (95%) was treated with ammonium hydroxide solution. added, followed by 1 ml of sodium The yellow fluorescence indicated the hydroxide (40%). The pink colour in ethanol presence of flavonoids. layer indicated presence of proteins. Detection of coumarines Detection of amino acids 10% NaOH (1 ml) was added to 1 ml of the For each plant extract, 1 ml of NaOH (20 ) plant extracts. The formation of yellow was added to 1 ml of the filtrate. An orange colour indicated presence of coumarines. colouration indicated the presence of amino acids. Detection of saponins

Detection of phytosterols (triterpenoides) Distilled water (2 ml) was added to each plant extracts and shaken. Formation of 1 The Liberdmann-Burchard test was used to cm foam indicated the presence of saponins. test for the presence of phytosterols. Five mg of each plant extract were each dissolved Detection of quinones in 2 ml of acetic anhydride and one or two drops of concentrated sulphuric acid was Concentrated sulphuric acid (1 ml) was added slowly along the sides of the test tube. added to 1 ml of each of the plant extract.

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Formation of red colour indicated the which was used as the negative control, presence of quinones. showed no activity. Hence, any inhibitions observed in the plant extracts were not due Detection of cardiac glycosides to the solvent. An inhibition zone diameter of 11 mm was chosen as a break-point of Glacial acetic acid (2 ml) and few drops of bacterial susceptibility of the extracts and 5% ferric chloride were added to 0.5 ml of the antibiotic (CLSI, 2014). The sensitivity each extract. Then, 1 ml of concentrated of E. coli to the various plant extracts was sulphuric acid was added. Formation of classified as being resistant, intermediate or brown ring at the interface indicated sensitive (Table 1). presence of cardiac glycosides. Minimum Inhibitory Concentration Detection of terpenoids (MIC) of plants extracts against E. coli

Chloroform (2 ml) and 1 ml of concentrated The MIC for each plant extract was sulphuric acid were added carefully to 0.5 determined. The results show that C. molle ml of extract. Formation of red brown colour and C. fragrans extracts did not inhibit the at the interface indicated the presence of growth of the bacteria at a concentration of terpenoids. 0.313 mg/ml, while Combretum micranthum extract did not inhibit the growth of the Data analysis bacteria at a concentration of 1.25 mg/ml (Table 2). C. molle and C. fragrans extracts All the experiments were run in triplicate. inhibited growth at a concentration of 0.625 Data were recorded and analyzed, using mg/ml, while C. micranthum extract SPSS 19.0 software. Comparison between inhibited growth at a concentration of 2.50 the main parameters was made by variances mg/ml. These values were taken as the MIC analysis (One-way ANOVA), at the 0.05 of the extracts. Acetone, on the other hand, level. did not inhibit the growth of E. coli.

Results and Discussion Qualitative phytochemical analysis of plants Antimicrobial assay of plant extracts Preliminary phytochemical analysis of the The results of antibacterial sensitivity of plant extracts indicated the presence of acetone extracts of C. molle, C. fragrans and different phytochemicals (Table 3). The C. micranthum by well diffusion method are extracts of C. micranthum and C. fragrans depicted in table 1. These results revealed contained high amounts of alkaloids which that all extracts are potent antimicrobials were completely absent in C. molle extracts. against studied E. coli. Varying levels of Proteins and tannins were present in trace antibacterial activity were observed with amounts in C. molle extracts. Terpenoides zones of inhibition diameters ranging from were totally absent in extracts of C. 08 ± 0.35 mm, for acetone extracts of C. fragrans. Carbohydrates and saponins were micranthum to 15 ± 1 mm, for extracts of C. completely absent in all the three plant molle. Ciprofloxacin, which was used as a extracts, while quinones were present only positive control, had a zone of inhibition in extracts of C. micranthum. All the plants diameter of 36 ± 1.80 mm and acetone, in this study contained tannins.

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translocation, phosphorylation steps and According to the growth inhibition zone other enzyme-dependent reactions diameters, all the used plants extracts (Seenivasan et al., 2006). These showed different levels of antibacterial phytochemicals also have some strong activity against tested E. coli. The obtained antimicrobial significance against some growth inhibition zone diameter of potential enteric pathogens (Edeoga et al., Ciprofloxacin is in accordance with the 30- 2005). 40 mm CASFM (2014) values, validating, thus, our test. The variation ranged in the Many of the traditionally used medicinal order of Combretum molle > Combretum plants have been investigated scientifically fragrans > Combretum micranthum (p < for antimicrobial activity and a large number 0.05). Previous studies, on plants extracts of plant products have been shown to inhibit antimicrobial activities show that diameters growth of pathogenic bacteria (Palombo et of growth zone inhibition vary with the al., 2006). The results in the present study plants species, the polarity of extraction show that the test isolate of E. coli is solvent, the test concentrations of the plants susceptible to extracts of C. molle and C. extracts, the extracted part of the plant and fragrans while C. micranthum showed the bacterial strains (de Morais Lima et al., mitigated bioactivity against E. coli. Many 2012; Kokora et al., 2013; Banfi et al., food borne pathogens have developed 2014). The varied antibacterial activity, resistance to antimicrobial agents, including observed in this study, may be due to the E. coli strains (White et al., 2002). This presence of different types of bacterium is known to be drug-resistant phytochemicals in varying concentrations. (O Ryan et al., 2005). It has been suggested Tannins, flavonoids, alkaloids, saponins, that this resistance to plant extracts may be reducing sugars, sterols and triterpenes have due to the presence of the outer membrane been reported to have antidiarrhoeal as well of the bacterial cell wall, which acts as a as antibacterial activity (Lewis, 2003; barrier to various environmental factors such Palombo, 2006). For instance, flavonoids as antibiotics or due to the differences in the have an ability to inhibit intestinal motility cell wall composition of various bacteria and hydro-electrolytic secretion, inhibit the (Tiwari et al., 2005). intestinal secretory response induced by prostaglandin E2 and have antioxidant The small zone of inhibition shown by C. properties responsible for the inhibitory micranthum indicates that although some effects exerted upon several enzymes plants are used in the management of (Venkat et al., 2006). Elsewhere, the diarrhoea, not all prescribed antidiarrhoeal antidiarrhoeal activity of the phytochemical medicinal plants may be very effective compounds has been attributed to their against enteric pathogens. However, this antimicrobial activity (Mamatha, 2006). intermediate position does not rule out the Terpenoides were totally absent in C. potential of the plant as antibacterial agents fragrans extracts but found in extracts of C. for treatment of enterobacterial infections. molle and C. micranthum. Earlier studies on The weak activity demonstrated by extracts the effects of the terpenoides on isolated of C. micranthum in vitro, does not bacterial membranes reveal their site of necessarily mean that the plant would action to be at the phospholipids bilayer. demonstrate weak activity in vivo. As, with They affect bacterial processes that include some drugs, some of these plant extracts the inhibition of electron transport, protein may be more potent in vivo due to metabolic

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 transformation of their components into Ocimum gratissimum inhibit the growth of highly active intermediates and to immune E. coli, Klebsiella pneumoniae, Bacillus adjustment (Ndip et al., 2009). subtilis and Salmonella typhimurium at 100 mg/ml, while Proteus vulgaris, Results of the minimum inhibitory Pseudomonas aeruginosa and concentration revealed that E. coli is Staphylococcus aureus are inhibited at 50 resistant to C. molle, and C. fragrans mg /ml. In the same study it is borne out that extracts at 0.313 mg/ml, while it is resistant extract of Myristica fragrans has the MIC of to C. micranthum extract at 1.25 mg/ml. The 100 mg/ml for Bacillus subtilis, Salmonella MIC for C. molle and C. fragrans is 0.625 typhimurium and Pseudomonas aeruginosa, mg/ml, confirming their strong antibacterial 50 mg/ml for E. coli, Klebsiella properties, while that of C. micranthum is pneumoniae, Proteus vulgaris and 25 mg/ml 2.5 mg/ml. Varying extracts of the studied for Staphylococcus aureus. The Combretum species have been found to Antibacterial activity (MIC) of the acetone inhibit E. coli growth at different MIC extracts of the stem bark of C. molle against values. For instance, acetone extract of the Helicobacter pylori 252C (clinical isolate) is bark of C. molle, ethanol leaves extracts of shown to be 2.5 mg/ml (Nyenje, 2011). C. micranthum, ethanolic extract of the stem Also, MIC of 2.5 mg/ml has been obtained bark of C. micranthum and methanol on the clinical strain of E. coli, with aqueous extracts of C. molle inhibit E. coli growth, plant extracts of Terminalia mantaly respectively, at 50 mg/ml (Asres et al., (Kokora et al., 2013). These results are 2006), 1.25 mg/ml (Banfi et al., 2014), 0.23 comprehensive, because most plants of the mg/ml (Agboke et al., 2012) and 2.5 mg/ml family are known to contain (Saidu, and Abdullahi, 2011). Moreover, the antimicrobial compounds (Baba-Moussa et MIC of acetone extract of the leaves of C. al., 1999). nioroense, acetone extract of the leaves of C. acutum, acetone leaves extract of C. Phytochemical screening is one of the paniculatum and methanol leaves extract of necessary steps to find out the chemical C. calobotrys, for E. Coli, have been found constituents which lead to the isolation of to be, respectively, 20 mg/ml (Coulidiati et compounds. Many of these compounds are al., 2011), 0.625 mg/ml (Coulidiati et al., used as the active ingredients of the modern 2009), 12.5 mg/l (Mbajiuka et al., 2014) and medicine or as the lead compounds for new 4.30 mg/l (Eziké et al., 2011). Besides, drug discovery. Phytochemical analysis of Elegami et al., (2007) find that the MIC of the acetone plant extracts of C. molle, C. methanol extracts of leaves of C. fragrans and C. micranthum revealed the adenogonium, C. glutinosum and C. presence of varied chemical components. aculeatum are of 4.69, 9.38 and 2.35 mg/l They are known to show medicinal activity for E. coli. The discrepancy between these as well as exhibiting physiological activity results can be related to the plants species, and exhibit anti-inflammatory, anti-oxidant their extracted part, and their geographical and membrane stabilizing property (El- localization, which acts on their chemical Mahmood, 2009). Many phytochemical contents, the extraction solvent and the studies of the stem bark of C. molle lead to tested bacteria species. Many other plants of the isolation of triterpenoides, glycosides, the Combretaceae family have been found tannins, alkaloids, saponins, stilbenes, to have antibacterial activity. Ibrahim et al., triterpenes saponin-oleanones 2011 have demonstrated that the extracts of trypetitepenes, arjunolic and mollic acids and glycosides which demonstrate cytotoxic,

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 antifungal, antimicrobial and anti- type 1 reverse transcriptase (Asres et al., inflammatory activity, as well as anti-HIV 2001).

Table.1 Diameters of growth inhibition zones of plants extracts, acetone (solvent) and Ciprofloxacin against E. coli

Plant extract Diameter of inhibition Susceptibility zone (mm) Combretum fragrans 12 ± 0.85 Sensitive Combretum micranthum 08 ± 0.35 Intermediate Combretum molle 15 ± 1.00 Sensitive Ciprofloxacin (positive control) 36 ± 1.80 Sensitive Acetone (negative control) 0 Resistant <7 = Resistant, 8-10 = Intermediate, 11 = Sensitive

Table.2 Minimum Inhibitory Concentration of plants extracts against E. coli

Plant extracts Concentrations (mg/ml) MIC (mg/ml) 0.078 0.156 0.313 0.625 1.25 2.50 5 10 Combretum fragrans G G G NG NG NG NG NG 0.625 Combretum G G G G G NG NG NG 2.50 micranthum Combretum molle G G G NG NG NG NG NG 0.625 Acetone G G G G G G G G Key: G = Growth, NG = No Growth.

Table.3 Profile of phytochemicals present in plants extracts

Tested chemical group Plants extracts Combretum fragrans Combretum micranthum Combretum molle Alkaloids ++ ++ _ Aromatic amino acids ++ ++ ++ Carbohydrates _ _ _ Cardiac glycosides ++ ++ ++ Coumarines ++ ++ ++ Flavonoids ++ ++ ++ Glycosides ++ ++ ++ Phenols ++ ++ + Proteins ++ ++ + Quinones _ ++ _ Saponins _ _ _ Tannins ++ ++ + Terpenoides _ ++ ++ Key: - : Absent; +: Trace; ++: Present in appreciable quantity

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Extracts of C. fragrans and C. micranthum against E. coli. The results from these had the highest amount of tannins. Tannins investigations shows that C. molle was the have been known to be effective in the most active plant, among the three plant prevention of colonization of enteric extracts, against E. coli, with the highest pathogens and consequently control zone of inhibition diameter. This study also diarrhoea (Palombo, 2006). These revealed the presence of various substances also precipitate proteins of the phytochemical compounds with antibacterial erythrocytes, reduce peristaltic movement properties in the three plants extracts. and intestinal secretion (Venkat et al., Varying levels of activity were observed 2006). Tannins are found in large quantities within the medicinal plants under study. It in the bark of trees where they act as a may be due to the quality and quantity of the barrier for micro-organisms like bacteria and active compound present in each plant fungi. They have been found to form extract. irreversible complexes with proline rich protein, resulting in the inhibition of cell E. coli is developing resistance to commonly protein synthesis (Shimada, 2006). Herbs employed antibiotics and is a common cause that have tannins as their main components of diarrhoea. Therefore, the plant extracts in are astringent in nature and are used for this study can be used in the treatment of E. treating inflamed or ulcerated tissues, coli generated diarrhoeal diseases. intestinal disorders such as diarrhoea and dysentery (Parekh and Chanda, 2007). Although this study has provided useful data Similar mechanisms of action could be concerning the antimicrobial activities of responsible for the antimicrobial actions of leaves extracts of C. molle, C. fragrans and the plant extracts under study. In another C. micranthum further works are necessary study, Asres et al. (2001) demonstrates good to provide more data. For instance, studies activity of the acetone extract of C. molle involving a large number of resistant stem bark against Mycobacterium pathogens that cause diarrhoeal diseases are tuberculosis and Plasmodium falciparum necessary to draw meaningful conclusions; 3D7. The activity has been attributed to high in vivo studies in order to confirm the amount of hydrolysable tannins present in antibacterial activity results obtained must the stem bark of the plant. It is generally be carried out; deep phytochemical analysis believed that tannins are non-selective of the plants extracts should be performed to enzyme inhibitors due to their polyphenolic isolate and characterize the specific groups. However, it has been shown that compounds responsible for the antibacterial some hydrolysable tannins display selective activity; the plant extracts should be cytotoxicity (Asres et al., 2001). screened for toxicity in vivo, in order to be able to determine the safest dosage for the In conclusion, plants are studied as potential treatment of diarrhoea in humans. disease controlling agents in humans as they are relatively safer, affordable and are easily Acknowledgements accessible at a local level, such that they can offer an alternative treatment option to the The authors acknowledge the Social conventional antibiotics. This study has Insurance Fund Hospital of Maroua allowed us to determine the antibacterial (Cameroon), for the provision of laboratory properties of the acetone leaves extracts of space where this work was done, the C. molle, C. fragrans and C. micranthum laboratory staff, for their advices and

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 399-411 technical support and the Annex of Garoua compounds from the stem bark of of the Pasteur Centre of Cameroon, for Combretum molle. Phyt. Res., 15: 613 providing them with E. coli isolate needed to 617. carry out this research work. Asres, K., Mazumder, A. Bucar, F. 2006, Antibacterial and antifungal activities of References extracts of Combretum molle. Ethiop. Med. J., 44: 269 77. Acharyya, S., Amarendra, P. Prasanta, K. Baba Moussa, F., Akpagana, K. Bouchet, P. 2009. Evaluation of antimicrobial 1999. Antifungical activities of seven activity of some medicinal plants West African Combretaceae used in against enteric bacteria with particular traditional medicine, J. reference to multi-drug resistant Vibrio Ethnopharmacol., 66: 335 338. cholera. Trop. J. pharm. Res., 8: 231 Banfi, S., Caruso, E., Orlandi, V., Barbieri, P., 237. Cavallari, S., Vigano, P., Clerici, P. Adegboye, M.F., Akinpelu, D.A. Okoh, A.I. Chiodaroli, L. 2014. Antibacterial 2008. The bioactive and phytochemical activity of leaf extracts from properties of Garcinia kola (Heckel) Combretum micranthum and Guiera seed extract on some pathogens. Afri. J. senegalensis (Combretaceae), Res. J. Biotechnol., 7: 3934 3938. Microbiol., 9: 66 81. Adesokan, A.A, Akanji, M.A. Yakubu, M.T. Basile, A., Sorbo, S., Giordano, S., Ricciardi, 2007. Antibacterial potentials of L., Ferrara, S., Montesano, D., Castaldo aqueous extract of Enantia chlorantha Cobianchi, R., Vuotto, M.L. Ferrara, L. stem bark. Afri. J. Biotechnol.,. 6: 2502 2000. Antibacterial andallelopathic 2505. activity of extract from Castanea Agboke, A.A., Udobi, C.E. Effiong, U.O. sativaleaves. Fitoterapia, 71: 110 116. 2012. Antibacterial potentials of the Bebora, L.C., Oundo, J.O. Yamamoto, H. ethanolic extract of the stem bark of 1994. Marked resistance of Escherichia Combretum micranthum G. Don and its coli strains, recovered from chickens, to fractions. J. Plant Stud., 1: 75 81. antibodies with particular reference to Ahmad, I. Aqil, F. 2006. In vitro efficacy of Trimethoprim-sulfamethoxazole bioactive extracts of 15 medicinal plants (Septrin). East Afri. Med. J., 71: 624 against ES L-producing multidrug- 627. resistant enteric bacteria. Microbiol. CASFM (Comité de l Antibiogramme de la Res., 162: 264 275. Société Française de Microbiologie. Akanga, J.O. 2008. Screening of antidiarrhoea 2014. Recommandations 2014, SFM, medicinal plants for in vitro France, 59 Pp. antimicrobial activity against clinical CLSI (Clinical and Laboratory Standard and environmental enteropathogens. Institute). 2014. Performance standards M.Sc. Thesis, Jomo Kenyatta for antimicrobial susceptibility testing, University, Nairobi, Kenya. twenty-fourth informational supplement. Akinpelu, D.A. Kolawole, D.O. 2004. Document M100 S24, 34, 230 Pp. Phytochemical and antimicrobial Coates, A, Hu, Y., Bax, R. Page, C. 2002. The activity of leaf extract of Piliostigma future challenges facing the thonningii (Schum.). Sci. Focus J., 7: development of new antimicrobial 64 70. drugs. Nat. Rev. Drug. Disc., 1: 895 Asres, K, Bucar, F., Knauder, E., Yardley, V., 910. Collins, C.H, Lynes, P.M. Grange, J.M. 1995. Kendrick, H. Croft, S.L. 2001. In vitro th antiprotozoal activity of extract and Microbiological methods, 7 edn.

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